A growing body of evidence suggests that release of glutamate (Glu), aspartate (Asp) and other excitatory amino acids (EAAS) play important roles in many disease processes of the mammalian CNS (see reviews by Rothman and Olney, 1986; Cotman et al., 1989), including ischemia (Simon et al., 1984), hypoglycemia (Weiloch, 1985), epilepsy (Nadler et al., 1978), Huntington's disease (Coyle and Schwartz, 1976) and Alzheimer's disease (Maragos et al., 1987). The concept that neuronal damage is mediated through the release of EAAs and subsequent stimulation of EAA receptors is of great clinical importance (Meldrum, 1985), including the development of therapy with drugs that decrease EAA release. Although much has been leamed in recent years about the toxicity of EAAs to neurons (Choi et al.,1988), the processes producing the release of EAAs remain largely unknown. The goal of this proposal is to test the following hypotheses: At least two pools of EAAs in the CNS can be released independently during metabolic insults. These pools differ in their location, the calcium-dependence of their release and also the degree of ATP depletion required to produce their release. Pool 1 is released from neurons in a Ca-independent manner by reversal of the Na-EAA cotransporter during severe metabolic insults that perturb Na+ concentrations. Pool 2 is selectively released from terminals in a Ca-dependent manner during mild ATP depletion. To test these hypotheses we will accomplish the following abbreviated Specific Aims: 1. To test that Pool 1 is released in a Ca-independent manner from neurons by reversal of the Na-EAA cotransporter, we will show that reversal of the cotransporter by either (i) replacement of extracellular Na+ or (ii) inhibition of the Na/K ATPase with ouabain will increase extracellular EAAs while decreasing intracellular EAAS. We will determine whether severe ATP depletion releases EAAs in a Ca-independent manner consistent with reversal of the Na-EAA cotransporter. 2. To test that Pool 2 is released from terminals during less severe ATP depletion, we will confirm that mild ATP depletion results in a Ca-dependent release, that mild ATP depletion selectively decreases terminal Glu compared to non-terminal Glu, and that mild ATP depletion produces a rise of intracellular Ca++ correlated with release of terminal Glu. 3. To examine the independence of the two pools, slices and cultures enriched for neurons and glia will be exposed to conditions that selectively release EAAs from each pool to determine if the combined treatment causes inereased release.